Research Overview

The major ongoing research projects in our lab right now are:

1. Replication and expansion of simple DNA repeats
2. Transcription-replication interplay and its effect on the organization and stability of the genome
3. Unusual DNA structures, including DNA triplexes

1. Replication and expansion of simple DNA repeats

Uncontrollable expansions of trinucleotide repeats lead to more than two dozen human hereditary neurological disorders, including Fragile X mental retardation, Huntington's disease, myotonic dystrophy, Friedreich's ataxia, etc. The molecular mechanisms of repeat expansions have, therefore, attracted a very broad attention. Our lab is pursuing a hypothesis that abnormal replication of expandable repeats could be in charge of this phenomenon. Using two-dimensional electrophoretic analysis of the replication intermediates, we were the first ones to demonstrate that replication fork is indeed stalled within those repetitive runs in a length and orientation-dependent manner in vivo. While our original observations were made in a model bacterial system, we have subsequently extended them into eukaryotic cells, including yeast and mammals. In all three systems, expandable repeats attenuated DNA replication. There was a good agreement between the repeats’ lengths, causing replication blockage in our systems, and their expansion thresholds in human pedigrees. Furthermore, there was a clear-cut correlation between the strength of the replication stalling and the repeat’s propensity to expand/contract in our experimental system. Finally, specific mutations in the replication proteins drastically increased the frequency of repeat expansions. Based on these observations and many supporting data from other labs, we have proposed a replication model for repeat expansions. It implies that the replication fork stalling at expandable repeats is caused by their ability to form stable DNA structures in the lagging strand template. Expansions and contractions supposedly occur during the imprecise replication fork restart within those repetitive runs, as presented below.

 

We are currently pursuing these studies in several directions. We analyze the replication of other structure-prone repeats, differing from trinucleotide repeats, to affirm that replication stalling is the universal phenomenon for this class of DNA sequences. We have recently developed an experimental system, which allows us to select for the large-scale expansions and/or contractions of various repeats in yeast. We plan to develop a principally similar selection system in mammalian cells. These systems should help us to unravel the role of cis- and trans-acting factors in repeat expansions. In the long run, they could also help searching for drugs that affect the rates of expansions or contractions, which could be useful for treatment of the debilitating disorders, caused by expandable repeats. Finally, the experiments are under way to establish a link between the replication stalling and chromosomal fragility in mammalian cells.

2. Transcription-replication interplay and its effect on the organization and stability of the genome

Since transcription and replication share the same template, occasional collisions between the two machineries are inevitable and can interfere with both processes. We have recently found that the head-on collisions with elongating RNA polymerase is much more detrimental for the replication fork progression in vivo than the co-directional collisions. Furthermore, we have proven that these collisions are caused by the direct physical interaction of the two machineries, rather than the long-range alterations of the DNA template. These results, combined with the data on the preferred co-directional alignment of transcription units with the direction of replication in prokaryotes, have led us to suggest that the main disadvantage of the head-on collisions could be in their inhibitory effect on DNA replication.

Besides collisions with elongating RNA polymerases, we study the effects of the transcription initiation or termination complexes on the replication fork progression. This could be even more important, since most genes are not actively transcribed during DNA replication. We have recently found that the steadfast transcription initiation complexes inhibit the replication fork progression in an orientation-dependent manner, during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, co-directional orientation. Notably in both instances, the replication fork is stalled immediately after passing the coding region. Transcription regulatory signals, thus, serve as “punctuation marks” for DNA replication in vivo by attenuating the replication fork progression, as it has traversed the coding areas. This attenuation could provide an extra time for the repair or recombination machineries to clear the coding areas off the newly acquired mutations.
This project is now developing in several directions. First, we are expanding our collision studies from the E. coli into yeast S. cerevisiae and, eventually, mammals. Second, we plan to experimentally determine mutation rates in the transcribed areas that are replicated head-on or co-directionally. This study will be carried out in yeast, using selectable genes driven by the S-phase-specific promoters. Finally, we are starting a major bioinformatics project, aimed at estimating the sequence divergence between genes in numerous bacterial genomes depending on their positioning relative to the direction of the replication.

3. Unusual DNA structures, including DNA triplexes

More than a decade ago, we have characterized an unusual three-stranded DNA structure - H-DNA, or triplex - formed by homopurine-homopyrimidine mirror repeats.



Little did we know at a time that one of those repeats, (GAA)n/(TTC)n, will be eventually implicated in the development of a hereditary human disorder - Friedreich’s ataxia. We have since found that formation of unusual DNA structures by H motifs during the DNA synthesis in vitro could block various DNA polymerases. Remarkably, the polymerase itself triggered the formation of an unusual DNA structure that subsequently inhibited it. Simple DNA repeats including, but not limited to H motifs were thus called “suicidal sequences” for the DNA polymerization. It has now become apparent that various DNA repeats could serve as suicidal motifs for the RNA polymerase, as well. Considerable efforts are currently being devoted to the detection of DNA triplexes and other unusual DNA structures inside living cells and elucidating their biological roles in norm and disease.